1. Field of the Invention
This invention relates to an electrophoresis medium membrane containing an aqueous polyacrylamide gel for use in determination of base sequences in nucleic acids such as a DNA and a RNA.
2. Description of the Prior Art
In the technique for determining base sequences in nucleic acids such as a DNA and a RNA according to the chemical degradation process, the dideoxy process or the like, slab electrophoresis using an electrophoresis medium membrane containing an aqueous polyacrylamide gel (hereinafter referred to as a polyacrylamide gel membrane or simply as a gel membrane) is indispensable. In recent years, electrophoresis analysis has come into wide use. Also, with the advances made in the dideoxy process, there has arisen a need for a polyacrylamide gel membrane capable of accurately separating up to a high molecular part of a fragment of a nucleic acid.
On the other hand, in the case where a fragment of a nucleic acid is electrophoretically separated based on a difference in its molecular weight for the base sequence determination of the nucleic acid and an ordinary polyacrylamide gel membrane having a constant thickness is used for this purpose, the band intervals of the separated fragment become wider for a low molecular part and narrower for a high molecular part. As a result, the separation of the high molecular part of the nucleic acid fragment is deteriorated. Accordingly, in order to achieve good separation performance uniformly over a wide molecular weight range from a low molecular part to a high molecular part, there has heretofore been used a polyacrylamide gel membrane (gradient gel membrane) having a gradient in the acrylamide concentration or a gradient in the buffer concentration along the direction of electrophoretic migration. For example, Japanese Unexamined Patent Publication No. 60(1985)-235819 (EP 0 159 694A, U.S. Pat. No. 4,704,198) discloses a process of and an apparatus for producing an electrophoresis medium membrane containing a polyacrylamide gel and having a gradient in the polyacrylamide concentration (gel concentration gradient or pore size gradient) by polymerizing and cross-linking a thin layer of an aqueous solution containing acrylamide and a cross-linking agent on a support surface by use of ionized radiation such as electron beams. The apparatus for carrying out the disclosed process and the method of controlling the electron beams or the like for forming the concentration gradient in the gel membrane are very complicated. In general, gradient gel membranes require much time for preparation and cannot always be prepared successfully because of low reproducibility of the concentration gradient. Thus the gradient gel membranes have the drawback that it is difficult to prepare many gel membranes having the gradient with good reproducibility.
On the other hand, in preparation of a gel membrane, the gel membrane is generally formed between two flat glass sheets, and it is difficult to impart a desired gradient in the membrane thickness to the gel membrane. For this reason, no gel membrane having the membrane thickness gradient has heretofore been used.
As mentioned above, it was technically difficult to provide the gel membrane at least with the gradient of the gel concentration or of the membrane thickness. Therefore, no attention has heretofore been given to the use of both the concentration gradient and the membrane thickness gradient. Also, it has generally been known that, in the case where the gel membrane is provided with the gradient of only one of the gel concentration or the membrane thickness, a complicated gradient curve is necessary in order to obtain good separation performance and for separation of the high molecular part of a nucleic acid. However, it was found that, with a gel membrane having both the concentration gradient and the membrane thickness gradient, large effects can be obtained by a combination of simple gradient curves (or gradient straight lines) of the concentration and the membrane thickness, respectively. Furthermore, the aqueous polyacrylamide gel exhibits a different extent of swelling, depending on a difference in the concentration. Therefore, with the gradient gel membrane having a complicated concentration gradient curve, the gel membrane is readily deformed at the time the gel membrane after being subjected to electrophoresis and carrying a separated nucleic acid fragment image is peeled from the support. However, it was found that, with a gel membrane having a simple concentration gradient curve (or a concentration gradient straight line), little deformation of the gel membrane arises at the time of peeling thereof after electrophoresis is carried out.